Bai R, Choe K, Ewell J B, Nguyen N Y, Hamel E
Science Applications International Corporation-Frederick, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
J Biol Chem. 1998 Apr 17;273(16):9894-7. doi: 10.1074/jbc.273.16.9894.
The alphabeta-tubulin heterodimer has two high affinity guanosine 5'-triphosphate binding sites, so that purified tubulin usually contains two molecules of bound guanosine nucleotide. Half this nucleotide is freely exchangeable with exogenous guanine nucleotide, and its binding site has been readily localized to the beta-subunit. The remaining nonexchangeable guanosine 5'-triphosphate can only be released from tubulin by denaturing the protein. We replaced the exchangeable site nucleotide of tubulin with 2'-deoxyguanosine 5'-diphosphate, exposed the resulting tubulin to ultraviolet light, degraded the protein, and isolated ribose-containing peptide derived from the nonexchangeable site. A large cyanogen bromide peptide was recovered, and its further degradation with endoproteinase Glu-C established that cysteine-295 of alpha-tubulin was the major reactive amino acid cross-linked to guanosine by ultraviolet irradiation.
αβ-微管蛋白异二聚体有两个高亲和力的鸟苷5'-三磷酸结合位点，因此纯化的微管蛋白通常含有两分子结合的鸟苷核苷酸。其中一半核苷酸可与外源鸟嘌呤核苷酸自由交换，其结合位点已很容易地定位到β亚基上。其余不可交换的鸟苷5'-三磷酸只能通过使蛋白质变性才能从微管蛋白中释放出来。我们用2'-脱氧鸟苷5'-二磷酸取代了微管蛋白的可交换位点核苷酸，将所得的微管蛋白暴露于紫外线下，降解蛋白质，并分离出源自不可交换位点的含核糖肽。回收了一个大的溴化氰肽，用内蛋白酶Glu-C对其进一步降解表明，α-微管蛋白的半胱氨酸-295是通过紫外线照射与鸟苷交联的主要反应性氨基酸。
